In a pilot study we showed that endothelial nitric oxide synthase (eNOS) was dynamically expressed during the course of stem cell differentiation into endothelial cells in vitro. The expression of eNOS was down-regulated at the beginning of stem cell differentiation. It was also found that stem cells lost their specific surface marker SSEA-1 within 24 hours of incubation with the NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME). The present study is proposed to test the hypothesis that down-regulation of eNOS expression is required for stem cells to differentiate into endothelial cells. The specific questions to be addressed in this study are as follows: 1) What is the role of the NO-cGMP signal transduction pathway in the maintenance of characteristics of stem cells? 2) Can NOS inhibitors promote the differentiation of stem cells into endothelial cells? 3) Is the loss of the stem cell surface marker SSEA-1 reversible after treatment with NOS inhibitors (i.e., can the cells recover their multipotent stem cell characteristics? 4) Can exogenous NO supplied by NO donors prevent the differentiation of stem cells? 5) Will stem cells transfected with the eNOS gene lose their capability to differentiate? 6) Can NOS inhibitors increase the number of circulating endothelial progenitor cells and facilitate re-endothelialization of the injured arterial wall in vivo? If down-regulation of eNOS protein expression is critical for the differentiation of stem cells in other cell lines, it is anticipated that NOS inhibitors such as L-NAME will accelerate the stem cell differentiation process. Conversely, we expect that a NO donor will prevent or delay the onset of stem cell differentiation. Similarly, stem cells transfected with the eNOS gene are expected to lose their capability to differentiate as long as the eNOS gene is expressed actively in the cells. In vivo studies will determine whether NOS inhibitors increase the number of circulating endothelial progenitor cells and facilitate the re-endothelialization at sites of vascular injury. The data from these studies will provide new information concerning the regulatory mechanisms for stem cell differentiation, and may help to develop new approaches to modulate the direction and rate of stem cell differentiation both in vivo and in vitro.